A particular challenge in the delivery of a gene by a viral vector for therapeutic purposes is the preparation and accurate characterization of clinical dosage forms. Total particle measurement can be made by such techniques as electron microscopy of viral preparations or measurement of total DNA by optical density at 260 nm of a sodium dodecyl sulfate (SDS) treated virus suspension. However, infectivity of a viral preparation, i.e., the number of infectious viral particles in a preparation of virus, is more challenging to accurately measure.
Traditionally, infectivity particles are measured in culture by a plaque-forming unit assay (pfu) that scores the number of viral plaques as a function of dilution. An alternative to the pfu assay is the tissue culture infective dose procedure (TCID.sub.50), which estimates infectivity as a function of intracellular staining for an antigen by direct immunofluorescence. The methods suffer from limitations including a high degree of inter-assay variability and are affected by such factors as virus replication status, vector characteristics, and virus-cell interactions.
More recently, flow cytometry or FACS (fluorescence-activated cell sorter) assays have been used to measure the number of infected cells in cell cultures infected at relatively high multiplicities of infection. For example, Saalmuller and Mettenleiter (J. Virol. Methods 44:99-108 (1993)) disclose the identification and quantitation of cells infected by recombinant pseudorabies virus mutants by the reaction of intracellular .beta.-galactosidase expressed during infection with recombinant viruses with a fluorogenic substrate, followed by detection of positive cells in flow cytometry. Morris et al. (Virology 197(1):339-48 (1993)) studied the process of productive and non-productive recombinant AcMNPV infection in cultured cells by immunostaining cells to detect the reporter CAT gene product.
The instant invention addresses the need for a more accurate method of quantitating infectious viral particles in a population.